Project Summary Inherited deficiencies of blood coagulation glycoproteins factor VIII (FVIII) or factor IX (FIX) result in the bleeding disorders hemophilia A and B, respectively. These individuals require intravenous infusions of exogenous factor to treat and prevent bleeding events. However 30% of patients with severe hemophilia A and 1-3% of patients with hemophilia B will form neutralizing antibodies called inhibitors against FVIII and FIX respectively. Although the immune response to FVIII and FIX are both dependent on CD4+ T cell responses, inhibitors to FIX can induce allergic IgE-mediated hypersensitivity responses and nephrotic syndrome due to immune complex deposition, which are rarely seen in patients with FVIII inhibitors. Antigen presenting cells (APCs), including dendritic cells (DCs), B cells, and macrophages, are essential for sensing antigens and directing adaptive immune responses towards immunity or tolerance. DCs have an enhanced ability to capture and process antigens for presentation on MHC complexes to nave T cells. There is limited understanding of the underlying mechanisms and critical cellular components that mediate innate immunity to FVIII and FIX. The main objective of this work is to characterize the role of DCs in the innate immune response to FVIII and FIX. This proposal consists of 3 specific aims: 1) Evaluate the role of conventional DCs in CD4+ T cell dependent- antibody formation in murine models of hemophilia A and B, 2) Identify the mechanisms of FVIII and FIX recognition and internalization by conventional DCs, and 3) Determine the properties of FVIII and FIX that mediate DC activation and co-stimulatory signaling. In Aim 1, T cell responses and antibody formation will be measured after targeted depletion of APC subsets in mice immunized with FVIII, FIX, or ovalbumin. The role of phagocytosis, macropinocytosis, and receptor-mediated endocytosis through C-type lectin receptors DCIR2 and Dectin-2 in FVIII and FIX uptake under normal conditions and with inflammation will be evaluated to determine the mechanism of factor recognition and internalization by DCs in Aim 2. In Aim 3, the effect of increasing concentrations of FVIII and FIX on DC activation in vitro will be measured. Additionally, the effect of inhibiting DC co-stimulatory signals on T cell activation, antibody responses, and complement activation in hemophilia A and B mice immunized by intravenous, subcutaneous, or intraperitoneal injections of FVIII and FIX will be evaluated. A fundamental understanding of these mechanisms will be important for evaluating immunologic changes in hemophilia patients during early factor exposure and for the development of strategies that prevent and eradicate inhibitors. The career development plan outlined will address training in immunology, molecular biology laboratory techniques, and translational research to ensure successful transition to research independence.